CRISPR/Cas9 system suitable for saccharomyces polygene knockout as well as construction method and application of CRISPR/Cas9 system

A construction method and gene knockout technology, applied in the field of genetic engineering, can solve the problem of low efficiency of multi-gene knockout, and achieve the effect of high knockout efficiency, low cost, and promotion of genetic engineering transformation.

Active Publication Date: 2021-04-13
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the current low efficiency of multi-gene knockout in yeast

Method used

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  • CRISPR/Cas9 system suitable for saccharomyces polygene knockout as well as construction method and application of CRISPR/Cas9 system
  • CRISPR/Cas9 system suitable for saccharomyces polygene knockout as well as construction method and application of CRISPR/Cas9 system
  • CRISPR/Cas9 system suitable for saccharomyces polygene knockout as well as construction method and application of CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of Saccharomyces cerevisiae BJ5464 multiple gene knockout vector

[0037] 1. Selection of knockout targets

[0038] (1) First search and obtain the pdr5 (Gene ID: 854324), pdr10 (GeneID: 854506), pdr15 (GeneID: 854506) and pdr15 (GeneID: 852015) gene sequence.

[0039](2) Using the CRISPR tool (http: / / crispr.dbcls.jp / ), upload the target sequence and analyze it to obtain all potential target sites of the target gene, that is, all downstream 20bp sequences containing NGG characteristic bases. The sequence with higher evaluation score and closer to the start codon ATG was selected as the gRNA knockout target.

[0040] 2. Construction of single knockout vector

[0041] Based on the CRISPR knockout vector p426-PSNR52-gRNA.CAN1.Y-TSUP4, three single gene knockout plasmids (single knockout plasmids): p426-Δpdr5, p426-Δpdr10 and p426-Δpdr15 were constructed. Taking the construction of the vector p426-Δpdr5 targeting the target gene pdr5 as an exampl...

Embodiment 2

[0078] Example 2: Knockout of S. cerevisiae BJ5464 toxin resistance gene:

[0079] 2.1 Preparation of yeast competent cells

[0080] The yeast transformation kit S.c.EasyComp Transformation Kit (Invitrogen) was used to prepare competent cells of the yeast strain S.cerevisiae BJ5464, and the specific method was as follows:

[0081] (1) Pick a single colony of yeast and inoculate it in 5mL YPD medium, culture it overnight at 30°C and 220rpm until the OD600 value is between 3.0-5.0;

[0082] (2) Dilute the bacteria: transfer the bacteria solution into 5mL YPD medium, make the initial OD600 value between 0.2-0.4, cultivate it at 30°C, 220rpm for about 4-6 hours, and its OD 600 The value reaches 0.6-1.0;

[0083] (3) Collect bacterial cells: put 5 mL of bacterial liquid in a 50 mL centrifuge tube, centrifuge at 4000 g for 5 min at room temperature, and discard the supernatant;

[0084] (4) Wash away the residual medium: Take 5mL Solution I (Wash solution) to resuspend the bacter...

Embodiment 3

[0092] Example 3: Research on the ability of Saccharomyces cerevisiae to resist exogenously added toxins

[0093] After knocking out the pdr5, pdr10 and pdr15 genes of Saccharomyces cerevisiae and obtaining the toxin-sensitive strain BJ5464-D-, it is necessary to investigate the tolerance of the strain to the addition of exogenous toxin DON. There are two main purposes: (1) verification After knocking out the three genes of the pdr series, does it reduce or weaken the ability of Saccharomyces cerevisiae to resist exogenous toxins? (2) To lay the foundation for selecting the appropriate toxin concentration and verifying the function of suspected anti-toxin genes in the later stage. Therefore, this study investigated the tolerance of Saccharomyces cerevisiae to DON before and after knockout.

[0094] On the YPD plate with 100 μM DON toxin concentration, the Saccharomyces cerevisiae colony was obviously sparse and blurred with the increase of the dilution gradient, and the OD va...

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Abstract

The invention discloses a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system suitable for saccharomyces polygene knockout as well as a construction method and application of the CRISPR/Cas9 system. The CRISPR/Cas9 vector suitable for saccharomyces cerevisiae polygene knockout is constructed, toxin resistance genes pdr5, pdr10 and pdr15 in the CRISPR/Cas9 vector are knocked out, and the antitoxic effect of toxin sensitive saccharomyces cerevisiae is verified through an antitoxic experiment. The invention provides a polygene knockout CRISPR/Cas9 system knockout method of saccharomyces and even other fungi, and provides a reliable model strain for verifying the antitoxic effect of other toxin resistance genes. According to the invention, the terminator is introduced into the homologous repair template in advance to realize gene inactivation, so that the polygene knockout method provided by the invention has the advantages of simple vector construction, high knockout efficiency, low cost and the like. The CRISPR/Cas9 system can be widely applied to polygene knockout of saccharomyces and filamentous fungi, thereby promoting genetic engineering modification of the saccharomyces and filamentous fungi.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a CRISPR / Cas9 system suitable for yeast multi-gene knockout and its construction method and application. Background technique [0002] Saccharomyces cerevisiae is a widely used single-cell eukaryotic cell factory, which has the advantages of easy culture, clear genetic background and convenient and efficient gene manipulation. Saccharomyces cerevisiae is the first eukaryotic organism to be completely sequenced, has the ability of post-translational modification of protein expression, and does not contain endotoxins. These advantages make Saccharomyces cerevisiae widely used in the industrial production of recombinant proteins. Due to their close genetic background, Saccharomyces cerevisiae is often used to synthesize secondary natural products of eukaryotic origin, or to identify single functional proteins. The production of Artemisinic acid, the precurso...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/81C12N9/22C12N1/19C12Q1/04C12R1/865
CPCC12N15/113C12N15/81C12N9/22C07K14/395C12Q1/04C12N2310/20C12N2800/102
Inventor 章卫民叶伟朱牧孜李赛妮岑由飞李浩华
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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